Although the transfusion of human blood for elective surgery and trauma has been practiced for some 200 years, only recently have the extent of infectious and immunological risks and hazards of transfusions become more fully recognized. Transfusions are known to provoke immunological responses. That is, the recipient may reject the transfused cell, or more seriously, the transfused cells can immunologically reject the recipient. Additionally, transfusions can introduce viral pathogens including HIV as well as a spectrum of hepatotrophic viruses.
It is well known that red blood cells deteriorate during storage at 4.degree. C. Recently, data have accumulated that show that the changes provoked by refrigerated storage are, in certain fundamental ways, similar to those found in normal red cell aging within the body. The extent and severity of these changes are directly related to the duration of refrigerated storage. Buoyant density increases and surface area decreases with accompanying vesiculation (loss of lipid-encapsulated vesicles from the surface of the red cell membrane). See, for example, Richard E. Waugh et al., "Rheologic Properties Of Senescent Erythrocytes: Loss Of Surface Area And Volume With Red Blood Cell Age," Blood 79, 1358 (1992). The vesicles formed at 4.degree. C. appear to lack cytoskeletal proteins, and may contain concentrations of hemoglobin that are to some extent lower than those found in the red blood cell cytoplasm. Moreover, the tendency of stored red blood cells to bind autologous IgG antibody directed against polymerized band 3 increases with refrigerated storage time as is the case for aging red blood cells in vivo. See, for example, Ken Ando et al., "Increased Susceptibility Of Stored Erythrocytes To Anti-Band 3 IgG Autoantibody Binding," Biochimica et Biophysica Acta 1178, 127 (1993), and Philip S. Low et al., "The Role Of Hemoglobin Denaturation and Band 3 Clustering In Red Blood Cell Aging," Science 227, 531 (1985). Thus, it has become clear that, as hemoglobin begins to denature in vivo as well as in the refrigerator, it forms hemichromes that cross-link the cytosolic domain of the major erythrocyte membrane-spanning protein, band 3, into clusters. The extracellular projections of the band 3 clusters comprise the extracellular antibody recognition sites for antibodies directed against senescent red blood cells. In vivo, such antibody binding to the senescent red blood cells helps to trigger their removal from the circulation. See, for example, Low et al., supra. Ordinarily, refrigerated red cells are outdated and discarded by the end of week six since, by this time, as many as one-quarter of such transfused cells are rapidly removed from the circulatory system by the spleen of the transfusion recipient. The loss of stored red blood cells as a consequence of outdating may exceed one million units of blood each year within the continental United States alone.
There is an additional compelling need to develop technology for safely extending the storage life of red blood cells. A would-be autologous (patient-to-self) donor cannot provide a unit of blood for storage more often than about once every two weeks without seriously depleting the body's existing red blood cell cadre. Because blood can be safely stored only for five to six weeks, by the time a patient is able to donate a third or fourth unit, the first unit collected would already have begun to deteriorate. Thus, in practice, an individual may safely donate three to four units of blood to his or her own account; however, there are many frequently performed surgical procedures that require more than 14 units of blood, especially those which require extracorporeal support for the circulation and oxygenation of blood.
Currently, red blood cells are stored in the presence of adenine and glucose using, in addition, a variety of soluble salts including such cations as magnesium, sodium, and potassium and such anions as chloride and citrate, which prevent hemolysis as well as cell clumping and coagulation of serum proteins. However, the refrigeration-associated changes including loss of membrane surface area, vesicle formation, and an increase in susceptibility to autologous antibody binding against band 3, as well as an increase in buoyant density that are mentioned above, are not adequately prevented by current practices and procedures.
Red blood cells are also stored in the frozen state, either in the presence of glycerol or in the presence of dimethylsulfoxide. See, for example, Charles E. Huggins, "Frozen Blood: Principles Of Practical Preservation," Monograph In The Surgical Sciences 3, 133 (1966). The use of dimethylsulfoxide has been banned in the United States because of its toxicity and because it also causes hemolysis as well as subtle damage to red blood cell membrane structures. Glycerol is currently used for freezing red blood cells, especially by the military. See, for example, C. R. Valeri et al., "The Safety And Therapeutic Effectiveness Of Human Red Cells Stored At -80.degree. C. For As Long As 21 Years," Transfusion 29, 429 (1989). However, methods employing glycerol are expensive, labor intensive, cumbersome, and are also associated with non-trivial red blood cell losses. Moreover, red blood cells that have been frozen in glycerol must be cleansed of this material after thawing by a slow, labor-intensive process, and have a relatively short useful half-life once thawed, during which time they must be transfused or discarded.
Hemin and hemichrome (decomposition products of hemoglobin) have been suspected of having the capability to function as cell membrane-damaging agents. Hemichrome is now known specifically to cause polymerization of the red cell anion channel (band 3) in vivo, resulting in disorganization of the red cell membrane cytoskeleton and loss of connections between the cytoskeletal fibers and the lipid bilayer. See, for example, Low et al., supra. Hemin (formed from hemichrome) and Fe.sup.3+, which is formed from Hemin, act as catalysts for oxygen radical formation and subsequent damage to hemoglobin, the membrane cytoskeleton, and the lipid bilayer.
It has recently become apparent that cumulative oxidative damage is a limitation on the useful storage life of refrigerated red blood cells. Much research has recently been focused on the design and incorporation of antioxidants into the storage medium. These include analogues of reduced glutathione, vitamin C, and vitamin E. Overall, the advantages provided by such additions have been found to be modest. See, for example, R. Blaine Moore et al., "Ascorbate Protects Against tert-Butyl Hydroperoxide Inhibition Of Erythrocyte Membrane Ca.sup.2+ +Mg.sup.2+ -ATPase," Arch. Biochem. and Biophys. 278, 416 (1990). Interestingly, in Tanya Repka et al., "Hydroxyl Radical Formation By Sickle Erythrocyte Membranes: Role Of Pathologic Iron Deposits And Cytoplasmic Reducing Agents," Am. Soc. Hematology 10, 2753 (1991 ), the authors find that administration of pharmacological amounts of ascorbic acid to sickle disease patients may be potentially hazardous due to adversely tipping the delicate antioxidant/pro-oxidant balance, since their results indicate that ascorbate can have both antioxidant and pro-oxidant effects.
There have also been a number of attempts to develop "blood substitutes" where hemoglobin can be delivered to patients in a stabilized form as a substitute for intact red blood cells. As an example, in Jen-Chang Hsia, "Pasteurizable Freeze-Driable Hemoglobin-Based Blood Substitute," U.S. Pat. No. 5,189,146, which issued on Feb. 23, 1993, hemoglobin is stabilized in its tetrameric form. This composition is prepared by diluting whole blood with isotonic NaCl solution and filtering it to separate the red blood cells from the plasma. The red blood cells are then washed in isotonic saline and lysed with hypotonic phosphate buffer. Particulate matter is separated from soluble hemoglobin by filtration. Cell-free oxyhemoglobin is converted to deoxyhemoglobin by applying a vacuum or by gas exchange in the presence of a reducing agent. The resulting deoxyhemoglobin is stabilized by the addition of a cross linker that stabilizes a specific conformation in monomeric or polymeric conjugates. The stabilized compound is then washed and concentrated, carbon monoxide is added, and the material is subsequently pasteurized at 60.degree. C. for 10 hours. Both liquid solutions and dry powders of CO-stabilized cross-linked hemoglobins were found to be quite stable to long term storage at moderate temperatures (for example, 56.degree. C. for 60 days).
Removal of CO under aseptic conditions is a prerequisite for use of cross-linked hemoglobins as a blood substitute. Hsia teaches that the CO-stabilized product can readily be photoconverted in the presence of oxygen to the oxygenated derivative oxy-hemoglobin which is suitable for transfusion. This material finds application as a blood plasma expander for use in acute emergencies, but has a circulatory half-life of only 4-5 hours.
In this context, the capacity of visible light to efficiently photolyze carbon monoxide hemoglobin derivatives has been known for many years. In Eraldo Antonini et al., "Hemoglobin And Myoglobin In Their Reactions With Ligands," Frontiers Of Biology, Vol. 21, North-Holland Publishing Company, Amsterdam-London (1971), the chemistry of carbon monoxide derivatives of hemoglobin and myoglobin is discussed in detail, including the photochemistry thereof.
Carbon monoxide has also been used to reduce the membrane damage that is induced in red blood cells by H.sub.2 O.sub.2. In J. McKenney et al., "Decreased In Vitro Survival Of Hydrogen Peroxide-Damaged Baboon Red Blood Cells," Blood 76, 206 (1990), the authors observed that exposure of human red blood cells to low concentrations of hydrogen peroxide in vitro results in membrane damage as manifested by the generation of spectrin-hemoglobin complexes, decreased red blood cell deformability, and cell surface alterations. Moreover, such exposures to H.sub.2 O.sub.2 lead to enhanced phagocytosis of the oxidized cells by monocytes. Prior treatment with carbon monoxide, however, completely inhibited cellular alterations induced by the H.sub.2 O.sub.2, and, in particular, the formation of the spectrin-hemoglobin complexes. Because of their biophysical and post-transfusion survival characteristics, baboon red blood cells are considered to be a reasonable surrogate for human red blood cells. McKenney et al. do not, however, suggest or demonstrate the efficacy of the application of hemoglobin/red blood cell stabilization by hemoglobin ligand such as CO for the purpose of extending the useful shelf-life of refrigerated red blood cells.
It is clear that prevention of the formation of hemichrome and its toxic derivatives is preferable to attempts to minimize the damage that these species are capable of producing, once they have formed. However, to date there have been no applications of hemoglobin/red blood cell stabilization techniques using hemoglobin-stabilizing ligands applied to prolonging the useful shelf-life of refrigerated red blood cells.
Accordingly, it is an object of the present invention to substantially prolong the survival of refrigerated red blood cells in a manner consistent with the practice of autologous transfusion.
Another object of the subject invention is to prolong the survival of refrigerated red blood cells without the necessity for complex or labor intensive procedures to generate transfusible samples.
Additional objects, advantages and novel features of the invention will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.